Methods for stimulating cells using nanoparticles and external field

ABSTRACT

This invention presents methods for targeting and killing types of cells or organisms using Magneto-Electric Nano-Particles under the control of an external magnetic field. A method was also presented for using Magneto-Electric Nano-Particles to stimulate or rejuvenate cells under an external magnetic field.

CROSS-REFERENCE TO RELATED APPLICATIONS

This non-provisional application is a Continuation-in-Part of U.S.patent application Ser. No. 15/578,218 filed Nov. 29, 2017, which is aNational Stage Application of International Application NoPCT/US2016/037619 filed Jun. 15, 2016, which claims the benefit of U.S.Provisional Application No. 62/181,936 filed on Jun. 19, 2015, theentire disclosures of which three prior applications are incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to a method for targeting or stimulatingtypes of cells or organisms using nanoparticles and an external field,and more specifically, using Magneto-Electric Nano-Particles (MENPs) andan external magnetic field.

BACKGROUND OF THE INVENTION

An important step in killing cancer cells is to bring and accumulatecancer killing agents to sites containing cancer cells, or morepreferably, bring them to the membrane of cancer cells. A type of suchcancer killing agents is various nano-particles possessing certainproperties or carrying cancer-killing drugs. An effective method toachieve this purpose is to coat the nano-particles with ligands orantibodies that only bind to the membrane of the cancer cells to betargeted. This method depends on the availability of ligands orantibodies matching the cancer cells to be targeted There is also anEnhanced Permeability and Retention (EPR) effect that preferablyaccumulates nano-particles at a cancer site than normal cells because ofthe increased blood flow to a cancer site, However, the EPR may notaccumulate enough nano-particles to reach the amount and density neededfor treatment.

It is known that cell functions and growth can be affected by electricsignals One desired effect is the stimulation or rejuvenation of skincells for a firmer or more youthful appearance. S Kavanagh et al (“Useof a neuromuscular electrical stimulation device for facial muscletoning a randomized, controlled trial”, Journal of Cosmetic Dermatology,2012 December; 11(4):261-6. doi: 10.1111/jocd 12007) showed that in a12-week trial, facial neuromuscular electrical stimulation (NMES) wasassociated with increased thickness of the zygomatic major muscle andsubjective improvements in facial attributes. Such NMES devices usepoint contact electrodes to apply the electric stimulation, as a result,uniform stimulation at cellular levels cannot be achieved. Also, EPRstrongly depends on the nano-particles' size and thus is limited torelatively large nano-particles.

There is an urgent need for killing viruses and antibiotic-resistantbacteria, and a method that is effective and applicable to a largevariety of viruses and bacteria is lacking.

There is no prior art that possess the functions of the embodimentspresented in this application.

SUMMARY OF THE INVENTION

One aspect of the present invention provides a method for stimulatingcells comprising administrating Magneto-Electric Nano-Particles (MENPs)to the cells; applying a magnetic field to the MENPs to generate anelectric field, and stimulating the cells with the electric field

The above features and advantages and other features and advantages ofthe present invention are readily apparent from the following detaileddescription of the best modes for carrying out the invention when takenin connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The present invention is illustrated by way of example, and not by wayof limitation, in the figures of the accompanying drawings and in whichlike reference numerals refer to similar elements. All the figures areschematic and generally only show parts which are necessary in order toelucidate the invention. For simplicity and clarity of illustration,elements shown in the figures and discussed below have not necessarilybeen drawn to scale. Well-known structures and devices are shown insimplified form, omitted, or merely suggested, in order to avoidunnecessarily obscuring the present invention.

FIG. 1 shows an embodiment for targeting cells that are less negativelycharged than surrounding cells.

FIG. 2 shows measurement of Zeta Potential of MENPs as a function ofmagnetic field in blood or other body fluid.

FIG. 3 shows the components and attachments of another embodimentimaging head

FIG. 4 shows a method for killing bacteria or viruses using AC-MENPs.

FIG. 5 shows a method for killing targeted bacteria or viruses after theAC-MENPs have bound to or have electroporated inside the targetedbacteria or viruses.

FIG. 6 shows a method for using AC-MENPs to deliver chemicals or drugsto killing bacteria or viruses.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Reference may now be made to the drawings wherein like numerals refer tolike parts throughout. Exemplary embodiments of the invention may now bedescribed. The exemplary embodiments are provided to illustrate aspectsof the invention and should not be construed as limiting the scope ofthe invention. When the exemplary embodiments are described withreference to block diagrams or flowcharts, each block represents both amethod step and an apparatus element for performing the method step.

Representative embodiments of the invention include, but are not limitedto, the following:

Embodiment #1: A method for targeting cells that are less negativelycharged than surrounding cells comprising injecting a solution ofnano-particles; applying for a period of time an external field thatwill increase the negative charges of the nano-particles so as toproduce an electric gradient field that pushes the nano-particles to theless negatively charged cells.

Embodiment #2: The method in Embodiment #1 wherein the nano-particlesare Magneto-Electric Nano-Particles (MENPs) and the external field is amagnetic field.

Embodiment #3 The method in Embodiment #2 wherein the MENPs is made witha basic structure of CoFe₂O₄—BaTiO₃ core-shell.

Embodiment #4: The method in Embodiment #3 wherein the nano-particlesare further surface functionalized by a compound.

Embodiment #5 The method in Embodiment #1 further comprising using anexternal field to guide and/or enhance the accumulation of thenano-particles into a site containing the less negatively charged cellsthan the surrounding more negatively charged cells.

Embodiment #6: The method in Embodiment #2 further comprising placing,injecting or implanting one or more permanent magnets or electro-magnetsin or near a cancer site, wherein the magnet(s) serves to attract MENPsto and increase accumulation of MENPs in the cancer site, as well as toincrease the negative charges of the MENPs to steer the MENPs towardsthe cancer cells.

Embodiment #7: The method in Embodiment #2 further comprising injectingor implanting wires, liquid capsules, injectable macro-particles orother injectable or implantable forms, made of permanent magneticmaterial into a solid tumor site that are deep inside the body or anorgan, wherein the magnetic field produced by these injected orimplanted magnetic sources serve to localize and accumulate the MENPs atcancer sites, and at the same time to enhance the electric fieldgradient to push MENPs to the cancer cells.

Embodiment #8: The method in Embodiment #2 further comprising using anapparatus formed into a shape and dimension to fit a target area orvolume that is made of or using permanent magnetic material and with oneor more electromagnets embedded in the apparatus to generate a constantmagnetic field and a changing magnetic field simultaneously.

Embodiment #9: The method in Embodiment #2 wherein the magnetic field isa changing filed generating a varying or pulsating force to push MENPstowards less negatively charged cells.

Embodiment #10: The method in Embodiment #9 wherein the changingmagnetic field is generated by driving a periodic or irregularalternating current through one or more electro-magnets, or by varyingthe position of one or more permanent magnets.

Embodiment #11. The method in Embodiment #10 further comprising changingthe period, pattern, magnitude and/or direction of the alternatingmagnetic field to achieve desired movements of the MENPs.

Embodiment #12: A method for targeting cells that are less negativelycharged than surrounding cells comprising injecting a solution ofnano-particles that are made of a chemical composition that produces ahighly negative surface charge or are coated or conjugated withmolecules that increase the negative surface charge when they are in ablood stream or body fluid which creates an electric force gradientfield that drives the nano-particles towards less negatively chargedcells.

Embodiment #13 A method for skin and appearance improvement comprisingapplying, a solution containing Magneto-Electric Nano-Particles (MENPs),wherein applying is either topically spreading on a skin area or gettingand spreading the MENP solution into deeper skin layers or muscles or acombination; and applying a magnetic field to the area for a period oftime to cause the MENPs to generate an electric field to stimulate thecells.

Embodiment #14: The method in Embodiment #13 wherein the magnetic fieldapplied is a changing filed that generates changing electric field onthe MENPs to deliver pulsating electric stimulations to the cells thatare near, attached to or surrounding the MENPs.

Embodiment #15: The method in Embodiment #13 further comprisingadjusting the frequency, magnitude and/or direction of the alternatingcurrent driving one or more electromagnets to generate the changingmagnetic field to achieve a desired electric stimulation effect.

Embodiment #16: The method in Embodiment #13 further comprising applyinga constant magnetic field using one or more permanent magnets orelectromagnets driven by direct current immediately after the MENPsolution is applied or simultaneously while the MENP solution is beingapplied to keep the MENPs within a local area where it is applied.

Embodiment #17 The method in Embodiment #13 further comprising applyinga constant magnetic field to provide a bias to keep the MENPs to thearea or close to the surface to where the solution is applied, andapplying a changing magnetic field to generate a changing electric fieldon the MENPs to deliver pulsating electric stimulations to the cellsnear, attached to or surrounding the MENPs.

Embodiment #18 The method in Embodiment #13 wherein applying a solutionof MENPs is done using a magnetic applicator to keep the MENPs to thearea or close to the surface to where the solution is applied.

Embodiment #19: The method in Embodiment #13 further comprising using aflexible facial mask or facial mold made with permanent magneticmaterial and/or embedded with an array of electromagnets, to generate anevenly distributed magnetic field.

Embodiment #20 The method in Embodiment #13 further comprising coatingor conjugating the MENPs with targeting agents that bind to the skin ormuscle cells to be stimulated.

Embodiment #21 A method for deep muscle or tissue toning comprisinginjecting a solution of MENPs into the muscle or tissue; and applying amagnetic field to the area or volume to cause the MENPs to generate anelectric field to stimulate the cells of the muscle or tissue.

Embodiment #22: The method in Embodiment #21 further comprising coatingor conjugating the MENPs with targeting agents that bind to the muscleor tissue cells to be stimulated.

Embodiment #23 A method for removing MENPs from the body comprisingapplying an apparatus that have a sufficiently strong magnetic field tothe areas with MENPs to attract the MENPs to the apparatus and thusremoving them from the body.

Embodiment #24: A method for removing MENPs from the body comprisingapplying an extraction solution that contains agents that binds to theMENPs; and washing the MENPs off together with the extraction solutionafter the MENPs are attracted to the surface.

Embodiment#25: A method for disrupting or killing bacteria or virusescomprising coating, binding or conjugating Magneto-ElectricNano-Particles (MENPs) with immunoglobulin, antibody mimetic, ligands,or other antibodies, molecules or proteins that bind to a targetedbacterium or virus to produce Antibody-Conjugated MENPs (AC-MENPs),applying, ingesting or injecting a solution containing AC-MENPs to anarea or intravenously into the blood stream and allowing the AC-MENPs tobind to the targeted bacteria or viruses; and applying a magnetic fieldto the area to cause the AC-MENPs to generate an electric field tointerrupt the function or kill the targeted bacteria or viruses.

Embodiment #26 The method in Embodiment #25 wherein the magnetic fieldis a constant filed produced by one or more permanent magnets.

Embodiment #27: The method in Embodiment #25 wherein the magnetic fieldis a changing filed as produced by electromagnets driven by varyingelectric currents.

Embodiment #28: The method in Embodiment #27 further comprisingadjusting the characteristics of the magnetic field, to generate anelectric field sufficient to interrupt the function, or kill thetargeted bacteria or viruses while at the same time, avoiding orminimize damages to surrounding normal cells.

Embodiment #29: The method in Embodiment #25 wherein the MENPs has aCoFe₂O₄—BaTiO₃ core-shell structure.

Embodiment #30: The method in Embodiment #25 further comprising coatingthe MENPs' surface with one or more layer of compound before coating orconjugating them with immunoglobulin, antibody mimetic, ligands, orother antibodies, molecules or proteins that bind to a targetedbacterium or virus to produce AC-MENPs.

Embodiment #31 The method in. Embodiment #25 further comprising afterthe AC-MENPs have bound to or have electroporated inside the targetedbacteria or virus, applying one or more of the following mechanisms tokill the bacteria or virus:

applying an external magnetic field to generate strong enough electricfield on the AC-MENPs to kill the bacteria or virus via the applicationof a sufficiently strong external magnetic field;

applying an alternating external magnetic field to induce an alternatingelectric field on the AC-MENPs whereas the strength and frequency of thefield is selected such that it disrupts the functions of the bacteria orviruses;

applying an alternating external magnetic field to induce mechanicalmotions of the AC-MENPs to disrupt the functions or to physically damagethe bacteria or viruses, causing them to die off; where the mechanicalmotions may include linear motion, slicing, collisions or vibrations, orcombinations thereof;

applying an alternating external magnetic field of a resonant frequencythat is modified by the interaction of AC-MENPs with thenano-environment to induce a ferromagnetic resonance in the AC-MENPs todisrupt or shut down the operation of the bacteria or viruses.

Embodiment #32: The method in. Embodiment #25 further comprising afterthe AC-MENPs have bound to or have electroporated inside the targetedbacteria or virus, applying an external constant magnetic field tomodify the resonant frequency of the AC-MENPs, and applying analternating external magnetic field at a frequency that causes aferromagnetic resonance in the AC-MENPs at or near the modified resonantfrequency to selectively disrupt the function of the bacteria orviruses.

Embodiment #33 The method in Embodiment #25 further comprising forlocalized treatment applying a first magnetic field externally toproduce higher concentration of AC-MENPs at and around a site or in anorgan or body part with high concentration with the targeted bacteria orviruses before applying a second magnetic field to cause the AC-MENPs togenerate an electric field to interrupt the function or kill thetargeted bacteria or viruses.

Embodiment #34: The method in Embodiment #25 further comprising choosingthe characteristics of the magnetic field so that it causes electricfield on the AC-MENPs bound to the targeted bacteria or viruses todisrupt or damage the targeted bacteria or viruses but does not causeother AC-MENPs that still remain in the body and unbound to targetedbacteria or viruses to harm healthy or untargeted cells.

Embodiment #35: The method in Embodiment #25 further comprising using awaiting period to give the body sufficient time to excrete most or allof the free AC-MENPs that did not bind to targeted bacteria or virusesout of the body before applying the magnetic field to cause damages tothe targeted bacteria or viruses.

Embodiment #36 A method for disrupting or killing bacteria or virusescomprising coating, binding or conjugating a drug or chemical that killsa targeted bacterium or virus to MENPs; coating, binding or conjugatingantibodies that bind to a bacterium or virus to the drug-coated MENPs toproduce Antibody-Conjugated Drug-Coated MENPs (AC-DC-MENPs); applying,ingesting or injecting a solution containing AC-DC-MENPs to an area orintravenously into the blood stream and allowing the AC-DC-MENPs to bindto the targeted bacteria or viruses 603; and applying a magnetic fieldto cause the AC-DC-MENPs to release the drug or chemical to interruptthe function or kill the targeted bacteria or viruses.

Embodiment #37 A method for making MENPs comprising depositing a thinfilm with the required properties via sputter deposition, evaporation,or another deposition technique; and using ion beam proximitylithography or imprint or another advanced lithography method to cut thethin films into MENPs of desired shapes and sizes.

Method for Targeting a Type of Cells Using Nanoparticles and ExternalField

It is an established fact that cell membrane exhibits a negative charge,and moreover, the membrane of cancer cells stores a significantly lessnegative charge than normal cells, as much as an order of magnitude lessnegative surface charge compared to normal cells. Therefore, ifnano-particles also exhibit negative charge, the electric field gradientforce established by these charges will push the nano-particles towardscancer cells. The stronger the negative charges on the nano-particles,the stronger the force that will push the nano-particles to cancercells, thus achieving the objective of targeting cancer cells whileavoiding normal cells.

One embodiment is a method for targeting cells that are less negativelycharged than surrounding cells, e.g., targeting cancer cells which areless negatively charged than normal cells, comprising injecting asolution of nano-particles 101; applying for a period of time anexternal field that will increase the negative charges of thenano-particles so as to produce an electric gradient field that pushesthe nano-particles to the less negatively charged cells 102, see FIG. 1.

One type of nano-particles that can be used in this embodiment isMagneto-Electric Nano-Particles (MENPs) and the external field is amagnetic field. When a magnetic field is applied to MENPs, the particlesproduce an electric polarization due to the magnetic-electric couplingproperty of the particles. This electric polarization increases thenegative charges of the MENPs. The increased negative charges on theMENPs and the more negatively charged normal cells create a non-zeroelectric-field gradient that pushes the MENPs away from the normal celland moving towards the less negatively charged cancer cells, effectivelyincreasing specificity of the targeting of the cancer cells by theMENPs.

One type of MENPs is made with a basic structure of CoFe₂O₄—BaTiO₃coreshell. One embodiment uses 30-nm MENPs synthesized from thefollowing steps: 1) 0.058 g of Co(NO₃)₂.6H₂O and 0.16 g of Fe(NO₃)₃.9H₂Oare dissolved in 15 mL of deionized (DI) water; 2) 5 mL of aqueoussolution containing 0.9 g of sodium borohydride and 0.2 g ofpolyvinylpyrrolidone is added at 120° C. for 12 hours to obtain CoFe₂O₄nanoparticles; 3) BaTiO₃ precursor solution is prepared by adding 30 mLof DI water containing 0.029 g of BaCO₃ and 0.1 g of citric acid to 30mL ethanolic solution containing 1 g of citric acid and 0.048 mL oftitanium (IV) isopropoxide; 4) As-prepared CoFe₂O₄ nanoparticles (0.1 g)is added to the 60 mL of BaTiO₃ precursor solution and sonicated for 120min, 5) The resulted dispersed nanoparticles is dried on hot plate at60° C. for 12 hours, while stirring at 200 rpm; 6) The obtained powderis heated at 780° C. for 5 hour in a box-furnace and cooled at 52° C.min⁻¹ to obtain 30 nm-sized CoFe₂O₄—BaTiO₃ coreshell MENPs. In anotherembodiment, the nano-particles are further surface functionalized by acompound, e.g., a 2-nm thick coating of glycerol mono-oleate (GMO) usingthe following steps: (i) GMO-MENPs is prepared by incubating 0.1 mg ofGMO with 5 mg of MENPs in 5 mL of PBS (pH 7.4) buffer for 12 hours; toachieve uniform surface modification, the solution is slowly agitatedduring incubation; (ii) The solution is centrifuged at 20000 rpm for 20min at 10° C. to remove excess GMO; (iii) The obtained pellet isre-suspended in ethyl acetate acetone (70:30) solution andre-centrifuged three times to obtain GMO-MENPs (iv) Surface-modifiedMENPs were lyophilized and stored at 4° C. until further use. Theparticle size distribution can be measured by a Zetasizer Nano seriesthat uses the standard dynamic light scattering (DLS) approach. Themeasured Zeta Potentials were −45±1.72 mV for non-functionalized MENPsand −41.6±0.26 mV for functionalized (with GMO) MENPs. When magneticfield is applied to the MENPs, the Zeta Potential can change toapproximately −50 mV and −57 mV at approximately 25 Oe and 100 Oerespectively, see 201 in FIG. 2.

Since the electric gradient field force is effective on the MENPs whenthe nano-particles are in the close proximity of the less negativelycharged cells, one embodiment further comprises using an external fieldto guide and/or enhance the accumulation of the nano-particles into asite containing the less negatively charged cells than the surroundingmore negatively charged cells. In one case, one or more permanentmagnets or electro-magnets can be placed, injected or implanted in ornear a cancer site, whereas the magnet(s) serves to attract MENPs to andincrease accumulation of MENPs in the cancer site, as well as toincrease the negative charges of the MENPs to steer the MENPs towardsthe cancer cells.

Wires, liquid capsules, injectable macro-particles (i.e., particles thatare larger than the nano-particles described above) or other injectableor implantable forms, made of permanent magnetic material can beinjected into solid tumor site that are deep inside the body or anorgan. The magnetic field produced by these sources serves to localizeand accumulate the MENPs at cancer sites, and at the same time, toenhance the electric field gradient to push MENPs to the cancer cells.

In another embodiment, a changing magnetic field is used instead of aconstant one to generate a varying or pulsating force to push MENPstowards less negatively charged cells The changing magnetic field can begenerated by driving a periodic or irregular alternating current throughone or more electro-magnets, e.g., a sine or square wave current, or byvarying the position of one or more permanent magnets, e.g., rolling,rotating or moving back and forth of permanent magnets. The period,pattern, magnitude and/or direction of the alternating magnetic fieldcan be changed to achieve desired movements of the MENPs.

One advantage of using MENPs in the above embodiments is that theincrease of negative charge can be controlled externally using amagnetic field, either using a constant magnetic field to cause aconstant increase of negative charge on the MENPs, or using analternating magnetic field of a chosen pattern to cause a pulsatingchange of the negative charge on the MENPs. Another advantage is thatmagnetic field can be used to increase the accumulation MENPs at atargeted site and/or prevent the spreading MENPs from a targeted site.In applications where these two advantages are not important, oneembodiment for targeting cells that are less negatively charged thansurrounding cells comprises injecting a solution of nano-particles thatpossess high levels of negative charge without requiring an externalfield Higher negative charges on nano-particles can be obtained bychanging the chemical composition of the nano-particles or coat orconjugate nano-particles with molecules that increase the negativecharge of the nano-particles when they are in a blood stream or bodyfluid When the nano-particles that possess high levels of negativecharge are in close proximity of the less negatively charged cancercells, the interaction of the nano-particles and the more negativelycharged normal cells create an electric force gradient field that drivesthe MENPs towards the cancer cells.

Constant (DC) magnetic field and changing (AC) magnetic field playdifferent roles in the embodiments using MENPs for target and killingcancer cells. In cases where both a DC magnetic field and an AC magneticfield are desired at the same time, one embodiment is an apparatusformed into a shape and dimension to fit a target area or volume that ismade of or using permanent magnetic material and with one or more, e.g.,an array of, electromagnets embedded in the apparatus to generate a DCmagnetic field and AC magnetic field simultaneously.

Method for Skin or Neuromuscular Electrical Stimulation at CellularLevel Using MENPs and Magnetic Field

One embodiment is a method for skin and appearance improvementcomprising applying a solution containing MENPs to an area 301, whereasapplying can be topically spreading on a skin area in the same fashionas skin moisturizer, applying into deeper skin layer usingmicroabrasion, microdermabrasion, micropuncture using superfine needs,subcutaneous injection or other methods that can spread the MENPsolution into deeper skin layers or muscles; and applying a magneticfield to the area for a period of time to cause the MENPs to generate anelectric field to stimulate the cells 302. Because the electric field islocal to each MENP and the MENPs are spread out in the area, theembodiment achieves electric stimulation at cellular level, as shown inFIG. 3.

In one embodiment, a changing (AC) magnetic field is applied to generatechanging electric field on the MENPs to deliver pulsating electricstimulations to the cells near, attached to or surrounding the MENPs.The frequency, magnitude and/or direction of the alternating currentdriving one or more electromagnets to generate the AC magnetic field canbe adjusted to achieve a desired electric stimulation effect.

When it is desired to keep the MENPs within a local area where it isapplied, a magnetic field, preferably a constant (DC) magnetic fieldusing one or more permanent magnets or electromagnets driven by directcurrent, is applied immediately after the MENP solution is applied orsimultaneously while the MENP solution is being applied. One way toapply a magnetic field to keep the MENPs to the area or close to thesurface to where the solution is applied is to use a magneticapplicator, e.g., a patch or mold made with permanent magnetic materialthat is applied immediately after the MENP solution is applied orsimultaneously while the MENP solution is being applied. In anotherembodiment, the DC magnetic field provides a bias to keep the MENPs tothe area or close to the surface to where the solution is applied, and achanging (AC) magnetic field is additionally applied to generate achanging electric field on the MENPs to deliver pulsating electricstimulations to the cells near, attached to or surrounding the MENPs.

One application of the above embodiments is for a treatment to firm ortone facial or neck appearance To apply the magnetic field evenly foreven electric stimulation and effect, one embodiment uses a flexiblefacial mask or facial mold made with permanent magnetic material and/orembedded with an array of electromagnets, to generate an evenlydistributed DC magnetic field, or AC magnetic field, or a DC magneticfield and AC magnetic field simultaneously.

The above embodiment can be generalized for deep muscle or tissue toningby injecting the MENPs into the muscle or tissue and apply magneticfield to the area or volume. Thus, one embodiment is a method for muscleor tissue toning comprising injecting a solution containing MENPs to amuscle or tissue area or volume; and applying a magnetic field to thearea or volume to cause the MENPs to generate an electric field tostimulate the cells of the muscle or tissue.

When it is desired to prevent MENPs from spreading to other parts of thebody, MENPs can be coated or conjugated with targeting agents, e.g.,molecules or proteins that bind to the cells to be stimulated, e.g.,bind to skin cells or muscle cells.

When it is desired to remove the MENPs from the body, an apparatus,e.g., mask, mold or roller, that have a sufficiently strong magneticfield can be applied to the areas with MENPs to attract the MENPs to theapparatus, thus removing them from the body. Another embodiment uses anextraction solution that contains agents that binds to the MENPs and canattract the MENPs to the surface and wash them off together with theextraction solution.

Method for Killing Viruses and Antibiotic-Resistance Bacteria UsingMENPs and Magnetic Field

In this application, an antibody can mean immunoglobulin, antibodymimetic, ligands, or other molecules or proteins that bind to a targetedbacterium or virus.

One embodiment is a method for killing bacteria or viruses comprisingcoating, binding or conjugating antibodies that bind to a bacterium orvirus to MENPs to produce Antibody-Conjugated MENPs (AC-MENPs) 401;applying, ingesting or injecting a solution containing AC-MENPs to anarea or intravenously into the blood stream and allowing the AC-MENPs tobind to the targeted bacteria or viruses 402; and applying a magneticfield to the area to cause the AC-MENPs to generate an electric field tointerrupt the function or kill the targeted bacteria or viruses 403, asshown in FIG. 4. The magnetic field can be constant as produced by oneor more permanent magnets, or changing as produced by electromagnetsdriven by varying electric currents under the control of amicrocontroller or other analog or digital control circuits.

Furthermore, the characteristics of the magnetic field, e.g., frequency,pattern, magnitude and direction, can be adjusted to generate anelectric field sufficient to interrupt the function or kill the targetedbacteria or viruses. At the same time, the characteristics of themagnetic field may be chosen to avoid or minimize damages to surroundingnormal cells.

One embodiment of MENPs is CoFe₂O₄—BaTiO₃ coreshell which can beprepared as in Corral-Flores et. al (Corral-Flores, V, Bueno-Baques, D &Ziolo, R Synthesis and characterization of novel CoFe₂O₄—BaTiO₃multiferroic core-shell-type nanostructures. Acta Mater. 58, 764-769,2010) In this procedure, CoFe₂O₄ particles are first prepared by thestandard hydrothermal method, with 0.058 g of Co(NO₃)₂6H₂O and 0.16 g ofFe(NO₃)₃.9H₂O dissolved in 15 ml of distill water and 0.2 g ofpolyvinylpyrrolidone dissolved in 5 ml of aqueous solution containing0.9 g of sodium borohydride at 120° C. for 12 hours. Then, precursorsolution of BaTiO₃ is prepared by mixing 30 ml of aqueous solutioncontaining 0.029 g of BaCO₃ and 0.1 g of citric acid with 30 ml ofethanolic solution containing 1 g of citric acid and 0.048 ml oftitanium (IV) isopropoxide. Coreshell CoFe₂O₄—BaTiO₃ MENPs are preparedby mixing 0.1 g of CoFe₂O₄ nanoparticles in the BaTiO3 precursorsolution and the mixture is sonicated for 2 hours Once the CoFe₂O₄nanoparticles are thoroughly dispersed, the mixture is dried on the hotplate at 60° C. overnight while continuously stirring. The dried powderis subjected to 780° C. for 5 hours in a furnace (CMF-1100) and cooledat 52° C. per minute to obtain the coreshell MENPs of ˜30 nm diameter.

In another embodiment, the MENPs' surface is coated with a layer ofcompound, e.g., glycerol monooleate (GMO). The GMO layer can be coatedby adding 1 mg of GMO to 5 mg of MENPs in 5 ml of the PBS buffer. Themixture is then incubated for 12 hours while being slowly rotated inorder to achieve uniform coating. Upon completion of the incubationprocess, the nanoparticles are centrifuged at 20,000 rpm for 20 minutesat 10° C. The pellet is washed in ethyl acetate acetone (70:30) solutionand re-centrifuged. The washing process is repeated thrice to completelyremove the excess unbound GMO. Finally, the obtained pellet islyophilized for 48 hours and stored for further use.

In one embodiment, antibodies are conjugated to the compound-coatedMENPs, e.g., GMO-MENPs, e.g., covalently attaching antibodies onto thecompound-coated MENPs' or GMO-MENPs' surface, e.g., following, theprotocol described by Kocbek et al (Kocbek, P., Obermajer, N., Cegnar,M., Kos, J. & Kristl, J. Targeting cancer cells using PLGA nanoparticlessurface modified with monoclonal antibody. J. Controlled Release 120,18-26, 2007). In one example, to covalently attach antibodies tocompound-coated MENPs, the nanoparticle surface is preliminarilyfunctionalized In the case of GMO-MENPs, 1 mg of GMO-MENPs are added to500 μl of the PBS buffer (pH 7.4). To this solution, 25 μl ofN-(3-Dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC) and25 μl of N-hydroxysuccinimide (NHS), at 1 mg/ml concentration in the PBSbuffer (pH 7.4) are added. The solution is incubated for 4 hours at roomtemperature while being stirred slowly. Then, the sample is centrifugedat 14,000 rpm for 10 minutes at 10° C. and the pellet is washed threetimes with 1 ml of the PBS buffer (pH 7.4).To bind antibodies to thefunctionalized MENPs, 10 μl of the antibodies (1 mg/ml) is added to thepellet along with 300 μl of the PBS buffer (pH 7.4). The solution isincubated for 2 hours while being rotated slowly and kept further at 4°C. overnight. The solution is centrifuged at 14,000 rpm for 10 minutesat 10° C. The pellet s washed thrice with 1 ml of the PBS buffer (pH7.4) to remove any excess antibody.

In one embodiment, after the AC-MENPs have bound to or haveelectroporated inside the targeted bacteria or virus, one or more of thefollowing mechanisms is applied to kill the bacteria or virus:

(A). Apply an external magnetic field to generate strong enough electricfield on the AC-MENPs to kill the bacteria or virus, e.g., localelectric fields of ≥1000 V/cm, which can be attained a few nanometersaway from AC-MENPs via the application of an external magnetic field of≥100 Oe;

(B). Apply an alternating external magnetic field to induce analternating electric field on the AC-MENPs whereas the strength andfrequency of the field is selected such that it disrupts the functionsof the bacteria or viruses, thus causes them to die off;

(C). Apply an alternating external magnetic field to generate heat onthe AC-MENPs to kill the bacteria or viruses whereas the strength andfrequency of the field is selected to generate sufficient heat to killthe bacteria or viruses without harming surrounding cells or tissues;and

(D). Apply an alternating external magnetic field to induce mechanicalmotions of the AC-MENPs to disrupt the functions or to physically damagethe bacteria or viruses, causing them to die off; where the mechanicalmotions may include linear motion, slicing, collisions or vibrations, orcombinations thereof.

In another embodiment, a ferromagnetic resonance strongly dependent onthe interaction of AC-MENPs with its nano-environment (in the proximityof a few nanometers away from the nano-particles) is used to selectivelydisrupt or shut down the operation of the bacteria or viruses, whenAC-MENPs are bound to or have electroporated the bacteria or viruses.The ferromagnetic resonance of AC-MENPs depends on the saturationmagnetization, which in turn, because of the magneto-electric (ME)effect, strongly depends on the electric fields that are associated withthe interaction of AC-MENPs with the nano-environment. As thenano-environment changes, so does the saturation magnetization andconsequently the ferromagnetic resonance frequency(ies) This resonantfrequency or set of resonant frequencies can be varied in a wide rangeby varying intrinsic properties, e.g. the magneto-crystalline anisotropyenergy and the exchange coupling constant, or extrinsic properties, e.g.the shape-induced anisotropy energy. In addition, the resonantfrequency(ies) can be controlled by application of an external DCmagnetic field. By specifically selecting the resonant frequencies,certain functions of cancer cells can be shut down with a relativelyhigh specificity on demand. For example, the microtubules responsiblefor cancer cell proliferation could be remotely controlled viaferromagnetic resonance of the AC-MENPs Namely, the resonant frequencyof AC-MENPs in the proximity (of 2 nm) of the microtubules changesbecause of the changes in the saturation magnetization. The saturationmagnetization change is due to the ME effect caused by the interactionof the AC-MENPs and the microtubules. An external AC magnetic field atthe new modified resonant frequency can then by applied to disrupt orcause damages to the bacteria or viruses.

The above mechanisms of targeted killing of bacteria or viruses usingAC-MENPs provide a new treatment that is non-toxic or low-toxic. Thesteps of a preferred embodiment, as shown in FIG. 5, comprise:

Step 1 (501) Injecting or ingesting AC-MENPs, via subcutaneous (SC),intraperitoneal (IP), or intravenous (IV) injection (including IVinjection or dripping using a catheter), or oral intake (01), or byother means;

Step 2 (optional, 502) Applying a first magnetic field externally toproduce higher concentration of AC-MENPs at and around a site or in anorgan or body part with high concentration with the targeted bacteria orviruses This step is optional and applicable to a disease site this islocalized, e.g., the site of infection or attack by the bacteria orviruses, and is skipped and not or less applicable when the bacteria orviruses are widely distributed, e.g., in the circulatory system); and

Step 3 (503): Applying a second magnetic field to induce the AC-MENPs togenerate one or more of the effects in (A) to (D) listed above todisrupt the function of the bacteria or viruses.

For a bacteria or virus infection that is localized, a localized secondmagnetic field this is confined to the disease site is applied For adisease in which the targeted bacteria or viruses are widelydistributed, a wide-area second magnetic field that covers a large bodyarea or the whole or most part of the body is applied so that bacteriaor viruses that are circulating in the body can be killed.

In one embodiment, the strength and/or frequency of the second magneticfield in Step 3 is chosen to cause the AC-MENPs to kill targetedbacteria or viruses but does not cause other AC-MENPs that still remainin the body and unbound to targeted bacteria or viruses to harm healthyor untargeted cells. In another embodiment, a sufficiently long waitingperiod is inserted between Steps 1 and 3 to give the body sufficienttime to excrete most or all of the free AC-MENPs that did not bind tobacteria or viruses out of the body. This reduces the risk of AC-MENPskilling healthy or untargeted cells and gives more freedom in selectingthe strength and/or frequency of the second magnetic field in Step 3 tokill the bacteria or virus.

Shape, size, ME coupling and other properties are important for theembodiments of this invention. One embodiment for making AC-MENPs with awide range of properties comprises first depositing a thin film with therequired properties via sputter deposition, evaporation, or anotherdeposition technique, and then using ion beam proximity lithography(IBL) or imprint or another advanced lithography method to “cut” thethin films into AC-MENPs of desired shapes and sizes.

In addition to cure diseases caused by bacteria and viruses, the aboveembodiments can also be used for skin peeling to reveal a more youthfulnew skin. In one embodiment, a solution of MENPs coated, bound orconjugated with proteins or molecules that bind to skin cells areapplied to the a skin area, e.g., face, a strong magnetic field isapplied to produce electric field that kills the cells in a surfacelayer of the skin. The dead layer will then peel off, and a new layer ofmore youthful looking skin cells grow.

It is known that drugs or chemicals coated, bound or conjugated to MENPscan be released by applying an external magnetic field, as shown by Nairet. al (M Nair, R Guduru, P Liang, J Hong, V Sagar and S Khizroev,“Externally controlled on-demand release of anti-HIV drug usingmagneto-electric nanoparticles as carriers”, Nature Communications 4,Article number: 1707, 2013).

Another embodiment is a method for killing bacteria or virusescomprising coating, binding or conjugating a drug or chemical that killsa targeted bacterium or virus to MENPs (drug-coated MENPs) 601; coating,binding or conjugating antibodies that bind to a bacterium or virus tothe drug-coated MENPs to produce Antibody-Conjugated Drug-Coated MENPs(AC-DC-MENPs) 602; applying, ingesting or injecting a solutioncontaining AC-DC-MENPs to an area or intravenously into the blood streamand allowing the AC-DC-MENPs to bind to the targeted bacteria or viruses603; and applying a magnetic field to cause the AC-DC-MENPs to releasethe drug or chemical to interrupt the function or kill the targetedbacteria or viruses 604, as shown in FIG. 6. The magnetic field forreleasing the chemical or drug can be constant as produced by one ormore permanent magnets, or alternating as produced by electromagnetsdriven by varying electric currents under the control of amicrocontroller or other analog or digital control circuits.

In one embodiment, AC-MENPs or AC-DC-MENPs is used topically for killingbacteria or viruses on the surface of skins or in skin pores, e.g., intreating bacterial acne. A solution of AC-MENPs or AC-DC-MENPs isapplied to the affected skin area, and a DC magnetic field, or a DC andan AC magnetic field is applied to kill the bacteria or viruses usingthe induced electric field and/or the release chemical or drug.

In the foregoing specification, embodiments of the present inventionhave been described with reference to numerous specific details that mayvary from implementation to implementation The specification anddrawings are, accordingly, to be regarded in an illustrative rather thana restrictive sense. The sole and exclusive indicator of the scope ofthe invention, and what is intended by the applicant to be the scope ofthe invention, is the literal and equivalent scope of the set of claimsthat issue from this application, in the specific form in which suchclaims issue, including any subsequent correction.

1. A method for stimulating cells comprising administratingMagneto-Electric Nano-Particles (MENPs) to the cells; applying amagnetic field to the MENPs to generate an electric field; andstimulating the cells with the electric field.
 2. The method accordingto claim 1, which is for skin and appearance improvement, and whichcomprises applying a solution containing the MENPs, wherein saidapplying is either topically spreading on a skin area or getting andspreading the MENP solution into deeper skin layers or muscles or acombination thereof; and applying a magnetic field to the area for aperiod of time to cause the MENPs to generate an electric field tostimulate the cells.
 3. The method according to claim 2, wherein themagnetic field applied is a changing filed that generates changingelectric field on the MENPs to deliver pulsating electric stimulationsto the cells that are near, attached to or surrounding the MENPs.
 4. Themethod according to claim 2, further comprising adjusting the frequency,magnitude and/or direction of the alternating current driving one ormore electromagnets to generate the changing magnetic field to achieve adesired electric stimulation effect.
 5. The method according to claim 2,further comprising applying a constant magnetic field using one or morepermanent magnets or electromagnets driven by direct current immediatelyafter the MENP solution is applied or simultaneously while the MENPsolution is being applied to keep the MENPs within a local area where itis applied.
 6. The method according to claim 2, further comprisingapplying a constant magnetic field to provide a bias to keep the MENPsto the area or close to the surface to where the solution is applied,and applying a changing magnetic field to generate a changing electricfield on the MENPs to deliver pulsating electric stimulations to thecells near, attached to or surrounding the MENPs.
 7. The methodaccording to claim 2, wherein applying a solution of MENPs is done usinga magnetic applicator to keep the MENPs to the area or close to thesurface to where the solution is applied.
 2. method according to claim2, further comprising using a flexible facial mask or facial mold madewith permanent magnetic material and/or embedded with an array ofelectromagnets, to generate an evenly distributed magnetic field.
 9. Themethod according to claim 2, further comprising coating or conjugatingthe MENPs with targeting agents that bind to the skin or muscle cells tobe stimulated.
 10. The method according to claim 1, which is for deepmuscle or tissue toning and which comprises injecting a solution ofMENPs into the muscle or tissue; and applying a magnetic field to thearea or volume to cause the MENPs to generate an electric field tostimulate the cells of the muscle or tissue.
 11. The method according toclaim 10, further comprising coating or conjugating the MENPs withtargeting agents that bind to the muscle or tissue cells to bestimulated.
 12. The method according to claim 1, further comprising astep of removing MEN Ps from a body.
 13. The method according to claim12, wherein the step of removing MENPs comprises applying an apparatusthat have a sufficiently strong magnetic field to the areas with MENPsto attract the MENPs to the apparatus and thus removing them from thebody.
 14. The method according to claim 12, wherein the step of removingMENPs comprises applying an extraction solution that contains agentsthat binds to the MENPs; and washing the MENPs off together with theextraction solution after the MENPs are attracted to the surface. 15.The method according to claim 1, further comprising preparing the MENPswith a method comprising depositing a thin film with the requiredproperties via sputter deposition, evaporation, or another depositiontechnique; and using ion beam proximity lithography or imprint oranother advanced lithography method to cut the thin films into MENPs ofdesired shapes and sizes.